Inhibition of Monoacylglycerol Lipase reduces Ventilator Induced Lung Injury in Mice
Poster #: 136
Session/Time: A
Author:
Ryan Keon Washington, BS
Mentor:
Nagaraja Nagre, PhD
Research Type: Basic Science
Abstract
INTRODUCTION:
Acute respiratory distress syndrome (ARDS) is a life threatening form of Acute Lung Injury (ALI), and is a common cause of respiratory failure in critically ill patients. Approximately 10 to 15% of patients in the ICU meet the criteria of ARDS. ARDS is characterized by a dysregulated immune response leading to the recruitment of neutrophils into the alveolar space, interstitial and alveolar oedema, and injury to epithelial and endothelial cells. Mechanical ventilation is a crucial, life saving intervention for patients with ARDS. However, mechanical ventilation can exacerbate alveolar lung injury, leading to the development of Ventilator Induced Lung Injury (VILI). The development of lung protective ventilation protocols has reduced the incidence of VILI over the years, but there is still a clinical need for alternative protective strategies. The Endocannabinoid System has a wide array of physiological roles. One of which is the attenuation of aberrant immune responses. Indeed, a previous study by the Nagre Lab indicated that Cannabinoid Receptor 2 (CB2R) activation could ameliorate Pseudomonas aeruginosa (PA) induced lung injury. The Endocannabinoid system is composed of Cannabinoid receptors, endocannabinoids which interact with those receptors, and enzymes that break down or synthesize endocannabinoids. Monoacylglycerol lipase (MAGL) is an enzyme that regulates the activity of the endocannabinoid (2-AG) which serves as an endogenous ligand for both CB1R and CB2R. MAGL hydrolyzes 2-AG, preventing the activation of both CB receptors, and releasing arachidonic acid (AA) which serves as a precursor for the synthesis of pro-inflammatory eicosanoids. In this study, we wanted to examine the role of endocannabinoid signalling in VILI. We utilized LEI-515, a reversible, peripherally restricted MAGL inhibitor to pharmacologically inhibit MAGL in a two-hit murine lung injury model consisting of LPS induced inflammatory injury and high-tidal volume mechanical ventilation
METHODS:
Lipopolysaccharide (LPS) was intratracheally administered to C57BL/6J mice. 24 hours later, the mice received high tidal volume ventilation (30 mL/ kg bodyweight; 100 breaths per minute) for two hours. To inhibit MAGL, LEI-515 (10mg/kg) was administered intraperitoneally 1h after LPS exposure and 1h before ventilation. Control mice received normal ventilation (10 ml/kg body weight, 10 minutes). Bronchoalveolar Lavage Fluid (BALF) was collected and analyzed for cell number. Following this, BALF cells were differentially stained and analyzed for neutrophil percentage. BALF IL-1β level was assessed by ELISA. The lung injury was assessed by histology. NF-kB activation was analyzed via Western Blot.
RESULTS:
Inhibition of MAGL with LEI-515 significantly reduced the BALF total cell count, which was otherwise elevated by VILI. The LEI-515-treated mice also had a significantly lower percentage of neutrophils. Histological analysis (H&E staining) demonstrated mitigation of lung tissue injury in LEI-515-treated mice. Moreover, BALF IL-1β concentrations were substantially lower in the LEI-515 group compared to vehicle controls. Western blot analysis further revealed that LEI-515 suppressed VILI-induced NF-κB activation.
CONCLUSION:
In summary, these findings show that the inhibition of MAGL ameliorates acute lung injury induced by VILI in mice and MAGL could be a promising therapeutic target for ARDS.
Acute respiratory distress syndrome (ARDS) is a life threatening form of Acute Lung Injury (ALI), and is a common cause of respiratory failure in critically ill patients. Approximately 10 to 15% of patients in the ICU meet the criteria of ARDS. ARDS is characterized by a dysregulated immune response leading to the recruitment of neutrophils into the alveolar space, interstitial and alveolar oedema, and injury to epithelial and endothelial cells. Mechanical ventilation is a crucial, life saving intervention for patients with ARDS. However, mechanical ventilation can exacerbate alveolar lung injury, leading to the development of Ventilator Induced Lung Injury (VILI). The development of lung protective ventilation protocols has reduced the incidence of VILI over the years, but there is still a clinical need for alternative protective strategies. The Endocannabinoid System has a wide array of physiological roles. One of which is the attenuation of aberrant immune responses. Indeed, a previous study by the Nagre Lab indicated that Cannabinoid Receptor 2 (CB2R) activation could ameliorate Pseudomonas aeruginosa (PA) induced lung injury. The Endocannabinoid system is composed of Cannabinoid receptors, endocannabinoids which interact with those receptors, and enzymes that break down or synthesize endocannabinoids. Monoacylglycerol lipase (MAGL) is an enzyme that regulates the activity of the endocannabinoid (2-AG) which serves as an endogenous ligand for both CB1R and CB2R. MAGL hydrolyzes 2-AG, preventing the activation of both CB receptors, and releasing arachidonic acid (AA) which serves as a precursor for the synthesis of pro-inflammatory eicosanoids. In this study, we wanted to examine the role of endocannabinoid signalling in VILI. We utilized LEI-515, a reversible, peripherally restricted MAGL inhibitor to pharmacologically inhibit MAGL in a two-hit murine lung injury model consisting of LPS induced inflammatory injury and high-tidal volume mechanical ventilation
METHODS:
Lipopolysaccharide (LPS) was intratracheally administered to C57BL/6J mice. 24 hours later, the mice received high tidal volume ventilation (30 mL/ kg bodyweight; 100 breaths per minute) for two hours. To inhibit MAGL, LEI-515 (10mg/kg) was administered intraperitoneally 1h after LPS exposure and 1h before ventilation. Control mice received normal ventilation (10 ml/kg body weight, 10 minutes). Bronchoalveolar Lavage Fluid (BALF) was collected and analyzed for cell number. Following this, BALF cells were differentially stained and analyzed for neutrophil percentage. BALF IL-1β level was assessed by ELISA. The lung injury was assessed by histology. NF-kB activation was analyzed via Western Blot.
RESULTS:
Inhibition of MAGL with LEI-515 significantly reduced the BALF total cell count, which was otherwise elevated by VILI. The LEI-515-treated mice also had a significantly lower percentage of neutrophils. Histological analysis (H&E staining) demonstrated mitigation of lung tissue injury in LEI-515-treated mice. Moreover, BALF IL-1β concentrations were substantially lower in the LEI-515 group compared to vehicle controls. Western blot analysis further revealed that LEI-515 suppressed VILI-induced NF-κB activation.
CONCLUSION:
In summary, these findings show that the inhibition of MAGL ameliorates acute lung injury induced by VILI in mice and MAGL could be a promising therapeutic target for ARDS.